Vaccine antigens that direct immunity to conserved epitopes

ABSTRACT

A method of identification and elimination of immunodominant epitopes to elicit a response to secondary epitopes, especially conserved structures, is described, and applied to influenza haemagglutinin (HA). Identification of the primary epitopes in (HA), and replacement of amino acids having high LODrps with corresponding low LODrps amino acids produces an HA molecule which induces antibody responses to conserved HA residues. Modified HA molecules induce a broadly neutralizing vaccine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of PCT/US2011/064442, whichwas published in English as WO 2012/082634 on Jun. 21, 2012; and claimsthe benefit of U.S. Provisional Patent Application No. 61/457,028, filedDec. 13, 2010; and U.S. Provisional Patent Application No. 61/626,792,filed Oct. 3, 2011.

GOVERNMENT FUNDING

This invention was made with government support under AI081051 awardedby National Institutes of Health. The government has certain rights inthis invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 23, 2012, isnamed 08340403.txt and is 51,124 bytes in size.

BACKGROUND OF THE INVENTION

The present invention relates to a method of producing antigens thatelicit an immune response to conserved epitopes and is thereforeapplicable to pathogens for which the primary immune response isdirected at variable epitopes. Such a method is especially applicable toinfluenza vaccines. Accordingly, the invention also provides a universalvaccine against influenza.

A vaccine is designed to induce an immune response that recognizes apathogen (or pathogen virulence factors) and thereby prevents ormitigates disease. The choice of antigens is, therefore, important. Animmune response against surface exposed antigens is typically mosteffective against an infection. At the same time, because of this immuneresponse, such surface exposed antigens are under constant evolutionarypressure to evolve and evade the immune system. Thus, a vaccine thatelicits an immune response against a specific strain of pathogen may beextremely effective against that strain, but poorly effective againstvariant strains. To account for the evolution of virulent strains, thevaccine maker may therefore have to target multiple antigens, add newantigens as the pathogen evolves, or target conserved antigens.

A separate problem in vaccine design is that some epitopes elicit anundesirable immune response. For example, inducing non-neutralizingantibodies can enhance Fc-mediated infection of macrophages, which isthe mechanism behind Dengue shock syndrome. Another problem is theinduction of an immune response that cross reacts with host antigens.The most famous of these is Guillain-Barré syndrome which is associatedwith Campylobacter infection, but is also associated with influenzainfection. Guillain-Barré syndrome was a reported side-effect of the1976 swine flu vaccination program. Accordingly, the selection ofepitopes for vaccines is far from routine.

Influenza is well known for rapidly evolving different strains,requiring new vaccines every season. Influenza A causes seasonalepidemics affecting millions every year and resulting in the death ofbetween 250,000 and 500,000 people every year, with up to millions insome pandemic years, according to WHO. These seasonal epidemics andpandemics arise because of the constant evolution of the virus boththrough mutations (“antigenic drift”) and through genetic reassortmentthat occurs when two different influenza viruses infect the same cell(“antigenic shift”). Such reassortment is greatly enhanced by theability of influenza A to infect a variety of host species, includingbirds, humans, and other animals, notably pigs. Thus, recombinationbetween two or more viruses, with different primary hosts, may result innovel and highly pathogenic strains that are responsible for the greatinfluenza pandemics.

Among Avian H5N1 influenzas, for example, there is concern that ahuman-adapted H5 influenza virus will evolve by mutational (geneticdrift) and/or reassortment (genetic shift) mechanisms, to cause acatastrophic pandemic. It is believed that the virus that causes thepandemic will derive from H5 influenzas that are circulating in birdstoday, but differ from them in ways that are impossible to predict.Therefore, not only is there interest in producing vaccines against thecirculating strains of H5, there is also interest in developing vaccinesthat would not be restricted by inherent strain-specificity.

Such “universal vaccines” target conserved and evolutionarily stableviral epitopes, rather than the continuously changing hemagglutinin (HA)and neuraminidase (N) epitopes targeted by seasonal flu vaccines(Gerhard, W et al. Prospects for Universal Influenza Virus Vaccine.Emerging Infectious Diseases, 2006. 12: p. 569., Subbarao, K, et al.,Development of effective vaccines against pandemic influenza. Immunity,2006. 24(1): p. 5-9.). Universal flu vaccines to date have focused onthe highly conserved M2 and NP proteins (Kaiser, J., A One-Size-Fits-AllFlu Vaccine. Science, 2006, 312:380). However, M2 and NP proteins arenot abundant or easily accessible on the surface of infecting virionsand the immune responses to M2 and NP do not directly prevent infection.Thus, an antibody response against M2 and NP is greatly inferior to thatobtained by the standard seasonal influenza vaccine.

Haemagglutinin is abundant and surface exposed, and is a primary targetof the immune response against the standard influenza vaccine. However,the HA molecule is highly variant, and the immune response to HA isoverwhelmingly driven against the hypervariable regions of HA. Thus, intraditional influenza vaccination or natural infections, the protectivehumoral immune response is overwhelmingly directed at a limited numberof continuously evolving, strain-specific, primary antigenicdeterminants on the surface of the influenza hemagglutinin, and there isminimal cross reaction with or protection against other serotypes ofinfluenza. This creates a barrier to a “universal vaccine” asvaccination strategies are typically predicated on mimicking naturalprotective immunity.

There is now evidence of a weaker and more broadly protective type of“heterotypic” immunity, which is not based on the response to primaryantigenic determinants, but instead derives from responses to conservedviral antigens. It is now thought that heterotypic influenza protectiondoes occur at low levels in human populations.

For example, a heterosubtypic response to seasonal influenza vaccine canbe observed by isolating B-cells that produce antibodies that bind toconserved epitopes (Corti, D., et al., Heterosubtypic neutralizingantibodies are produced by individuals immunized with a seasonalinfluenza vaccine. J Clin Invest, 2010. 120(5): p. 1663-1673). Naturalinfection can induce heterosubtypic antibodies that are crossprotective, but only at very low titre (Sullivan, J. S., et al.,Heterosubtypic antiavian H5N1 influenza antibodies in intravenousimmunoglobulins from globally separate populations protect against H5N1infection in cell culture. J Mol Genet Med, 2009. 3(2): p. 217-24; seealso Sui, J., et al., Wide prevalence of heterosubtypic broadlyneutralizing human antiinfluenza A antibodies. Clin Infect Dis, 2011.52(8): p. 1003-1009; Wrammert, J., et al., Broadly crossreactiveantibodies dominate the human B cell response against 2009 pandemic H1N1influenza virus infection. J Exp Med, 2011. 208(1): p. 181-193).Epidemiological data collected before and during the 1957 flu pandemicsuggested that heterosubtypic immunity to HA may be observed in adultsbut not in children (Epstein, S., Prior H1N1 influenza infection andsusceptibility of Cleveland Family Study participants during the H2N2pandemic of 1957: an experiment of nature. J Infect Dis., 2006. 193: p.49-53.), and raises the possibility that elicitation of protectiveheterotypic responses may prove effective against avian influenzaviruses.

More recent studies have advanced the concept of “seasoned” immunity.Through multiple infections with different strains, a “seasoned”response to conserved epitopes may be observed. (Lynch, G. W., et al.,Seasoned adaptive antibody immunity for highly pathogenic pandemicinfluenza in humans. Immunol Cell Biol, 2011, pp 1-10, Wrammert et al. JExp Med, 2011. 208(1): p. 181-193). This response, while low, issufficient to provide some degree of protection from heterotypic andheterosubtypic infection, and explains the greater heterosubtypicimmunity observed in adults than in children. It is to be emphasizedthat the immunity offered by heterotypic and heterosubtypic immunity canbe observed as a lower morbidity, mortality and viral shedding, but itis far inferior to the homotypic immunity usually obtained by standardvaccination or infection.

Heterotypic immunity has also been demonstrated by passiveadministration of a monoclonal antibody (C179) that recognizes aconserved conformational epitope on the hemagglutinin stem consisting ofHA1 318-322 and HA2 47-58. C179 reduced the severity of illness anddeath rate in mice infected with H1, H2 or H5 influenzas (Okuno, Y., etal., A common neutralizing epitope conserved between the hemagglutininsof influenza A virus H1 and H2 strains. J. Virol., 1993. 67: p. 2552-8.;Okuno, Y., et al., Protection against the mouse-adapted A/FM/1/47 strainof influenza A virus in mice by a monoclonal antibody withcross-neutralizing activity among H1 and H2 strains. J. Virol., 1994.68: p. 517-20.; Smirnov, Y., et al., Prevention and treatment ofbronchopneumonia in mice caused by mouse-adapted variant of avian H5N2influenza A virus using monoclonal antibody against conserved epitope inthe HA stem region. Arch Virol., 2000. 145: p. 1733-41.).

Recent attempts to create a universal vaccine have focused on elicitingan immune response against the stem/stalk domain. For example, Steel etal. (Influenza virus vaccine based on the conserved hemagglutinin stalkdomain, mBio 1 (1): 1-9 (April 2010)) describes vaccination with a“headless” HA molecule to drive an immune response against the stalkdomain of HA. Wei et al. (Induction of broadly neutralizing H1N1influenza antibodies by vaccination, Science 329: 2060-2064 (27 Aug.2010, e-pub 15 Jul. 2010)) describes how immunization with a DNA vectorexpressing H1N1 HA and then boosting with H1N1 seasonal vaccine orreplication defective adenovirus 5 vector encoding HA stimulated theproduction of broadly neutralizing antibodies that recognize H1 fromdiverse H1 isolates, with some cross-neutralization of H3 and H5.Further analysis indicated that the immune response was directed againststem antigens. Other research in this area has also been reported.(Bommakanti et al., Design of an HA2based Escherichia coli expressedinfluenza immunogen that protects mice from pathogenic challenge. ProcNatl Acad Sci USA, 2010. 107(31): p. 13701-6; Wang et al. Vaccinationwith a synthetic peptide from the influenza virus hemagglutinin providesprotection against distinct viral subtypes. Proc Natl Acad Sci USA,2010. 107(44): p. 18979-84.)

However, such “stem” or “headless” vaccines miss other conservedepitopes, such as those that exist on the head (Khurana, Antigenicfingerprinting of H5N1 avian influenza using convalescent sera andmonoclonal antibodies reveals potential vaccine and diagnostic targets.PLoS Med, 2009. 6(4): p. e1000049; Krause et al., A broadly neutralizinghuman monoclonal antibody that recognizes a conserved, novel epitope onthe globular head of influenza H1N1 virus hemagglutinin. J Virol, 2011.pmid_21849447; Whittle, et al., Broadly neutralizing human antibody thatrecognizes the receptorbinding pocket of influenza virus hemagglutinin.Proc Natl Acad Sci USA, 2011. 108(34): p. 1421621; Yoshida, et al.,Crossprotective potential of a novel monoclonal antibody directedagainst antigenic site B of the hemagglutinin of influenza A viruses.PLoS Pathog, 2009. 5(3): p. e1000350). Antibodies to the head domainblock hemagglutination, and therefore should restrict access to thereceptor binding site, and therefore preventing infection viainterference with viron binding to host cell sialic acid receptors.

Another site outside the stem region is the cleavage site between theHA1 and HA2 domains of HA. This region is highly conserved betweeninfluenza A and B hemagglutinin precursors, and peptide conjugatevaccines with sequences from the highly conserved maturational HA1/HA2elicited broadly protective immune responses against lethal challengefrom other A and B influenzas (Bianchi, E., et al., Universal influenzaB vaccine based on the maturational cleavage site of the hemagglutininprecursor. J. Virol., 2005. 79: p. 7380-8., 14., Horvath, A., et al., Ahemagglutinin-based multipeptide construct elicits enhanced protectiveimmune response in mice against influenza A virus infection. ImmunolLett., 1998. 60: p. 127-36.).

Given that conserved epitopes that mediate broad neutralization arepresent on the HA head, as well as its stem, vaccine antigens comprisedof entire trimeric hemagglutinins, rather than only the stem, ormimetics of selected broadly neutralizing epitopes, should offer thegreatest opportunity of heterosubtypic protection.

The challenge of generating an immune response against the conservedepitopes on the head is that such conserved epitopes are structurallylinked to the variable regions that are antigenically dominant. Theimmunodominant regions cannot be merely removed, however.

Epitopes on the surface of proteins are almost always discontinuous andconformation dependent (Barlow D J, et al., Continuous and discontinuousprotein antigenic determinants, Nature 1986; 322:747-748). Therefore,merely deleting the immunodominant region alters the structure of thehead and thus the structure of the conserved epitope. By contrast,immunization against the stem region is less problematic because theentire stem may be used.

The challenge remains to generate through vaccination an immune responseagainst conserved antigens, that is at sufficient titre to offermeaningful protection.

SUMMARY OF THE INVENTION

The invention provides a method of reducing the immune response to anepitope while retaining protein structure, comprising: (a) identifyingamino acids high on the log odds relative propensity scale (LODrps, ameasure of the likelihood of an amino acid being part of an epitope) and(b) replacing at least one high LODrps amino acid with a low LODrpsamino acid. By reducing or ablating the immune response to aprimary/immunodominant epitope, the immune response is directed againstsecondary epitopes, including conserved epitopes that are weaklyimmunogenic.

In a related embodiment, the invention provides a method of making avaccine that elicits an immune response against conserved epitopes on aprotein antigen, comprising:

(a) identifying a primary immunodominant epitope in the antigen;

(b) replacing at least one high LODrps amino acid in the primaryimmunodominant epitope with a low LODrps amino acid therebysignificantly eliminating the antigenicity of the primary immunodominantepitope, to create a modified antigen; wherein the modified antigeninduces antibodies against conserved epitopes. In some embodiments, atleast one high LODrps amino acid from each primary immunodominantepitope is replaced with a low LODrps amino acid.

The method of invention is suitable for the manufacture of a vaccine. Inrelated embodiments, the vaccine is used for immunization against adisease by administration of an antigen as described herein. In someembodiments, the prime and boost antigens are different.

In other embodiments, the invention includes a modified protein antigenin which a primary immunodominant epitope in the native protein antigenis modified by replacement of at least one high LODrps amino acid with alow LODrps amino acid, thereby significantly eliminating theantigenicity of the primary immunodominant epitope. This embodiment maybe usefully applied to influenza antigens, such as HA

Identification of the primary epitopes in influenza haemagglutinin (HA),and replacement of amino acids high on the log odds ratio propensityscale (LODrps) with corresponding low LODrps amino acids produces an HAmolecule which induces antibody responses to conserved HA residues. Suchmodified HA molecules are suitable for a broadly neutralizing vaccineagainst influenza. Accordingly, the invention concerns an influenzahaemagglutinin antigen in which all primary epitopes are modified toreduce antigenicity. In some embodiments, the haemagglutinin antigen isan H5 haemagglutinin.

Representative modifications include at Pro125, Ser129, Glu131, Pro140,Gln142, Lys144, Ser145, Lys156, Lys157, Asn158, Thr160, Arg166, Asp187,and/or Lys193 (H3 numbering). In some embodiments, the invention is anHA having one, two, three, four, five, six, seven, eight, nine, ten orall of these residues modified. Relatedly, the invention includes aninfluenza haemagglutinin antigen having a sequence at least 90%, 95, 98and 99% identical with the HA portion found in any of SEQ ID NOs: 2-10.

Other suitable modifications may be grouped according to domains: d1(P140, Q142, K144); d2 (K156, K157, N158), d3 (E131), d4 (D187, K193)and d5 (P125, R166). In some embodiments, one to eleven of these aminoacids are mutated. In another embodiment, mutagenesis of all of d1-d5may require only a single mutation in each domain.

Amino acids are replaced with low LODrps amino acids, such as alanine orthreonine.

In related embodiments, the invention includes a vaccine, comprising oneor more modified haemagglutinins and a pharmaceutically acceptablecarrier. In some embodiments the haemagglutinins are proteins. Suchproteins may be administered directly, or attached to carrier such as avirus-like particle, incorporated into a replication-defective viralparticle or inactivated virus. In other embodiments, the vaccine is inthe form of a nucleic acid (DNA, RNA, etc) which is administered to asubject, whereupon the haemagglutinin is expressed. “DNA immunization”is well known in the art.

The invention also includes a method of making an influenza vaccine,comprising

(a) identifying the primary epitopes on the HA molecule;

(b) replacing high LODrps amino acids in one or more primary epitopewith low LODrps amino acids;

wherein the vaccine induces neutralizing antibodies that arecross-protective against distantly related HA molecules.

The invention also includes related methods of using the compositions ofthe invention. Accordingly, the invention includes a method ofimmunizing a subject, comprising administration of one or more doses ofa vaccine made as described herein. In related embodiments, the subjectis immunized with a composition comprising an antigen with an HA havingmodifications at Pro125, Ser 126, Ser129, Glu131, Pro140, Gln142,Lys144, Ser145, Lys156, Lys157, Asn158, Thr160, Arg166, Asp187, and/orLys193; an antigen with an HA having modifications at Ser126, Ser129,Glu131, Pro140, Gln142, Lys144, Ser145, Lys156, Thr160, Arg166, Asp187,and/or Lys193, or an influenza haemagglutinin antigen having a sequenceat least 90% identical with the HA portion found in any of SEQ ID NOs:2-10. An immunization protocol may include immunization with more thanone xHA together in a single dose, multiple doses, and multiple doseswith different xHAs in each dose. An immunization protocol may includeimmunization with xHA and an adjuvant. Adjuvants, in the presentcontext, include cytokines and other immunomodulatory molecules such asTLR (toll like receptor) agonists and their derivatives that stimulatethe immune response.

In other embodiments, the xHA molecules may be bound to a structure thatenhances the immune response, such as a virus-like particle, animmunostimulatory molecule (e.g. Tetanus toxin fragment), a dendrimer,and the like.

The HA molecules of the present invention are also suitable for theelicitation of antibodies that are broadly cross protective, includingpolyclonal and monoclonal antibodies. Such antibodies can provide“passive immunity” against infection and/or treatment of infectedindividuals. For example, such antibodies can be obtained, purified,concentrated, and stored. Accordingly, the invention includes methods ofobtaining antibodies against the modified HA molecules, and antibodiesobtained by immunization with such molecules.

The methods and compositions described herein are suitable forgeneration of an immune response against influenza viruses, especiallyinfluenza A, and are therefore used in a vaccine against influenza Ainfection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the 2.9 Å 2FKO.pdb structure (Stevens, J., et al.,Structure and receptor specificity of the hemagglutinin from an H5N1influenza virus. Science, 2006. 312: p. 404-10.) of the hemagglutininfrom the A/Viet/1203/04 H5 influenza virus, with its monomeric subunitsdrawn in 3 different shades of gray.

The membrane-distal “head” of the trimeric HA is at the top of theillustrated molecule, and contains receptor binding and primaryantigenic determinant structures. The membrane-proximal “stem” is at thebottom, and contains conserved fusion peptide and HA1/HA2 cleavage sitesequences.

To create xHA antigens and shift the immune response from “head” primaryantigenic determinants to conserved HA structures (includingfusion-peptide CR6261 epitopes (white) on the HA stem (Ekiert, D. C., G.Bhabha, et al. (2009). “Antibody recognition of a highly conservedinfluenza virus epitope.” Science 324(5924): 246-51.)), up to 11 highLODrps amino acids in primary antigenic determinant(s) (black) werereplaced with amino acids having lower Discotope LODrps values.

FIG. 2 illustrates the Drosophila Expression System (DES) (InVitrogen)construct for production of recombinant V1203 hemagglutinin and xHAproduction. FIG. 2 discloses “His.6” as SEQ ID NO: 12.

FIG. 3. Purified recombinant xHAs bearing mannosylated oligosaccharidesfor immune presentation a, Reduced and b, Non-reduced SDS-PAGE ofpurified xHAs (200 ng/lane) c, GNA lectin blot of duplicate of gel in a.GNA binds to terminal mannose d, Undigested (O) and PNGase F (P)digested xHAs. left, sypro red stained gel; right, GNA-stained blot.

FIG. 4. Sequences of the parent hemagglutinin and xHAs. FIG. 4 disclosesSEQ ID NOS 1-10, respectively, in order of appearance.

The xHA.s were expressed in the Drosophila Expression System(InVitrogen) using the pmtbipv5hisa vector as shown in FIG. 2. Expressedpolypeptides were comprised of: (1) the BIP signal sequence frompmtbipv5hisa, (2) followed by the dipeptide Arg-Ser from a Bgl2 linkersequence, (3) followed by HA0 encoding sequences incorporating (a)substitutions in primary antigenic determinant sites as indicated inTable 3, and (b) substitution of a “T” rather the “RRRKK” (SEQ ID NO:11)(R) sequence at the HA1/HA2 cleavage site of wildtype V 1203, (4)followed by a polypeptide encoding a thrombin cleavage site, foldonsequence, and hexa-histidine (SEQ ID NO: 12) sequence tag, after theKREEIS (SEQ ID NO: 13) sequence of HA2.

The expressed parental and xHA hemagglutinin sequences are shown withthe BIP signal sequence in three letter code, primary antigenicsubstitutions primary antigenic determinant substitutions indicated asbold underlined residues, and C-terminal thrombin-foldon-hexaHis (SEQ IDNO: 12) polypeptide in bold. Italics indicate Xba I-Kpn I fragment usedfor rapid construction of the xHA variants.

FIG. 5: Immunoassays to verify proper folding of recombinanthemagglutinins and primary antigenic determinant knock out in xHAs.

FIG. 6 A-D Polyclonal antisera to xHAs contain antibodies that competefor binding of a conserved fusion peptide-containing epitope on the HAstem. Panel A shows no competition by mouse non-immune serum (m NI, opencircles), and competitive, concentration-dependent reduction of 1F02binding following exposure to the positive control C179 mAb (solidtriangles). Each of the plots in the remaining B-D panels presents 1F02competition results from serum samples obtained at various stages duringthe immunization of a single animal with xHAs. The designation “pr”indicates antisera obtained after priming, “b1” and “b2” arerespectively antisera obtained after boosts 1 and 2, etc. The animals inpanel B were primed and boosted with xHA.par (10 ug/injection). Theanimals in panels C and D, were primed with xHA.4b, then repeatedlyboosted with xHA.5b; those in C received 10 ug doses of the xHAantigens, while those in D received 20 ug doses.

FIGS. 7A-7D: Hemagglutination inhibition (HAI) by antisera to xHAs.Further data is provided in Example 6.

Hemaggutination inhibition assays were based on the standard W.H.O. kitprotocol for Hemagglutination testing. Antisera were treated overnightat 37° C. with 3 volumes of receptor destroying enzyme (RDE, Denka),which was subsequently inactivated for 30 m at 56° C. Hemagglutinationmicroplate wells were loaded with 25 ul of RDE-treated samplesrepresenting overall 18, 36 and 72-fold dilutions of the antisera, orwith PBS. The four HA-pseudotyped lentiviruses used for FIG. 2 HAneutralization assays were employed as ‘antigens’ in the HA inhibitionassays. The LV antigen stocks were adjusted to 8 HA units/50 ul, and 25ul (4 HA units) added to the antiserum- and PBS-containing wells. After30 minutes of incubation, 50 ul of glutaraldehyde fixed 0.5% turkey RBCs(Fitzgerald) were added to the antiserum+LV incubations, and plates werephotographed 40 min later.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Because the primary immune response to influenza is directed againstcontinuously evolving primary antigenic determinant structures on thehemagglutinin, the inventor has constructed a series of HA antigens inwhich these primary epitopes have been modified to ablate immunogenicitybut keep overall structure of the HA. The resulting antigen molecule(s)thereby stimulate the production of antibodies against secondaryantigenic determinants, which are epitopes that do not map to theprimary antigenic determinants. Antibodies against conserved elementsare broadly neutralizing.

To reduce uncertainty, the following definitions are used throughout:

“About” is used as understood by the person of ordinary skill in thecontext of the variable to which “about” is applied. When in doubt,“about” indicates a variation of ±10% of the stated value.

“Haemagglutinin” or “HA” refers to the Influenza haemagglutinin protein.In certain embodiments, this is the influenza A haemagglutinin, andrecombinant variants thereof. In exemplary embodiments, thehaemagglutinin is H5, or derived from H5. The term is used to describe afamily of proteins, without regard to whether the protein actuallypossesses the property of hemagglutination. HAs are traditionallygrouped by serotyping, into H1, H2, H3, H4, H5 and the like. Theserotypes reflect antigenic and genetic variation. There is alsovariation within a given serotype, but this is less than betweenserotypes.

“xHA” refers to an HA for which one or more primary antigenicdeterminants are modified. “xHA.par” refers to the recombinant parentalcontrol hemagglutinin, which in the exemplary case is the HA fromA/Vietnam/1203/2004 with the describe modification of the HA1/HA2cleavage site.

“Antigenic determinants”, or “epitopes,” are structures recognized byantibodies and T-cell receptors of the immune system. Preferably, suchepitopes are antibody epitopes. In the case of hemagglutinin, a smallnumber of structures on the ectodomain (head) surface induce antibodiesmuch more readily than the rest of the molecule and are referred to asbeing “immunodominant”.

“Primary antigenic determinants” are synonymous with “immunodominantepitopes” and are those to which the immune response is primarilydirected. If expressed numerically, an immune response that is at leastone order of magnitude greater to a given epitope would indicate that itis immunodominant. In the context of influenza HA, those epitopes in thehighly variable regions of the HA head to which the immune systemnormally develops the strongest antibody response, and which arerecognized following infection or immunization with conventionalseasonal flu vaccines, are the “primary antigenic determinants.” Thesedeterminants in influenza are constantly evolving.

“Secondary antigenic determinants” are not immunodominant. In thecontext of the present invention, secondary antigenic determinants arethose epitopes that are recognized after ablation of the primaryantigenic determinants. Secondary antigenic determinants may be locatedin the conserved regions of the HA, which occur both on the HA head andstem. “Ablation” of antigenic determinants traditionally occurred bydeletion of the entire epitope but is meant here to indicate ablation ofantigenicity, by substitution of amino acids. Following from theimmunodominant terminology, Secondary antigenic determinants may also bereferred to as “immunorecessive.”

“Escape mutant” refers to a derivative influenza virus that does notbind to, and/or is not neutralized by, a particular antibody orantiserum. As used herein, “escape mutant” refers to a virus containinga mutation in an epitope targeted by a neutralizing antibody.

“LODrps” is the “log odds ratio propensity scale” which measures thenatural logarithm of the odds-ratio of a given amino acid to be presenton the antigen side of an antigen-antibody interaction, and is thereforea measure of the likelihood of being an epitope. A high LODrps valuemeans that the amino acid is over-represented in the set of structurallywell-defined epitopes, whereas under-represented amino acids have lowlog-odds ratio values. The term LODrps is described in greater detailbelow.

A “vaccine” describes a preparation designed to induce an immuneresponse that is protective against disease. In the present context, avaccine induces an immune response against influenza virus. A vaccinemay be prophylactic or preventative, given prior to or shortly afterexposure to an influenza virus; or therapeutic, given during infectionto boost the immune response or drive the response in a specificdirection. A vaccine does not have to induce a fully protective responsethat prevents all disease, as not all vaccines produce an immuneresponse in all people, and the strength and nature of the immuneresponse varies between people.

“Antibody” as used herein encompasses natural antibodies, chimeric andrecombinant antibodies, and antibody fragments, such as Fab, scFv, andthe like.

An “adjuvant” increases the immune response against an antigen withwhich it is presented. Adjuvants are known in the art and includealuminium hydroxide, monophosphoryl lipid A, oils, cytokines, toll likereceptor agonists, and the like.

Influenza Hemagglutinin Molecular Structure

HA is the major surface protein of the virus as well as the major viraltarget of neutralizing antibodies. The 2.9 Å 2FKO.pdb structure(Stevens, J., et al., Structure and receptor specificity of thehemagglutinin from an H5N1 influenza virus. Science, 2006. 312: p.404-10) of the hemagglutinin from the A/Viet/1203/04 H5 influenza virusis shown in FIG. 1. It is a homotrimer with a large head (ectodomain)comprised of prominent beta sheet structures, and a stem (stalk)composed of long alpha helices. The base of the stem anchors thehemagglutinin in the viral membrane (or cellular membrane prior tobudding), while its ectodomain head is exposed on the surface of thevirus. HA plays an essential role in infection and the viral life cycleby (i) presenting binding sites for cellular receptors on its ectodomainsurface and (ii) mediating fusion of viral and host-cell membranes topermit cellular entry of the genome-transcriptase complex followingendocytosis.

Monomer subunits of the hemagglutinin trimer are synthesized as largeHA0 precursor molecules, which are cleaved by host proteases at a sitein the stem to generate HA1 and HA2 fragments of about 300 and 200residues, respectively. The head is composed of HA1 sequences only,while the stem is a structurally complex structure containing entwinedHA1 and HA2 sequences.

Naturally contracted influenza infections and seasonal flu vaccineselicit antibodies which bind primary antigenic determinant epitopes onthe HA head. The primary antigenic determinants are located adjacent tothe receptor binding sites. Accordingly, antibodies to primary antigenicdeterminants are neutralizing and block infection by restricting hostcell sialic acid receptor access to the HA receptor binding site.However, the protective function of these antibodies is short-lived dueto rapid evolution of primary antigenic determinants under selectivepressure.

The primary antigenic determinants on HA have been identified bysequencing “escape mutants” selected with neutralizing antibodies, andcorrespond to hot spots of sequence variation during virus evolution(Stevens, J., et al., Structure and receptor specificity of thehemagglutinin from an H5N1 influenza virus. Science, 2006. 312: p.404-10., Kaverin, N., et al., Structure of antigenic sites on thehaemagglutinin molecule of H5 avian influenza virus and phenotypicvariation of escape mutants. J Gen Virol., 2002. 83: p. 2497-505.,Kaverin, N. V., et al., Epitope mapping of the hemagglutinin molecule ofa highly pathogenic H5N1 influenza virus by using monoclonal antibodies.J Virol, 2007. 81(23): p. 12911-7., Philpott, M., et al., Hemagglutininmutations related to attenuation and altered cell tropism of avirulentavian influenza A virus. J. Virol., 1990. 64: p. 2941-7., Wiley, D., I.Wilson, and J. Skehel, Structural identification of the antibody-bindingsites of Hong Kong influenza haemagglutinin and their involvement inantigenic variation. Nature, 1981. 289: p. 373-8.). FIG. 1 shows thelocations (black) of H5 HA primary antigenic determinants identifiedusing avian H5 escape mutant data and sequence information from human H5drift isolates obtained during 1997-2004. The primary antigenicdeterminants surround and overlap the receptor binding domain (RBD)(Stevens, J., et al., Structure and receptor specificity of thehemagglutinin from an H5N1 influenza virus. Science, 2006. 312: p.404-10., Weis, W., et al., Structure of the influenza virushaemagglutinin complexed with its receptor, sialic acid. Nature, 1988.333: p. 426-31.), which mediates binding of the virus to the cell duringinfection. A RBD and adjacent primary antigenic determinants are presenton each monomer of the HA trimer.

In addition to the well known, narrowly-focused antibodies to thecontinuously evolving primary antigenic determinants on the HA head,there are also broadly-protective antibodies that recognizeevolutionarily conserved and functionally critical structures located onHA stem and head surfaces.

For example, mAbs C179, CR6261, F10 and 1F02 (see, e.g., Okuno J.Virol., 1993. 67: p. 2552-8; Ekiert et al. Science. 324 (2009); SuiVirology 387: 473-481 (2009); Wrammert et al. J Exp Med, 2011. 208(1):181-93) recognize fusion peptide-containing HA stem epitopes that areconserved in Group 1 influenzas, which include the H1, H2, H5 subtypes.Similarly, mAb CR8020 recognizes a fusion peptide-containing HA stemepitope conserved in Group 2 influenzas, which includes the H3 and H7subtypes (Ekiert et al. Science. 2011. 333:843-50). Finally mAb FI6recognizes fusion peptide-containing HA stem epitopes from both Group 1and Group 2 viruses (Corti, D. et al., Science, 2011. 333:850-6).

In addition to the broadly-protective antibodies recognizing conservedfusion-peptide containing structures located on HA stem, there are alsobroadly neutralizing antibodies that recognize conserved structures onthe HA head. (Khurana, Antigenic fingerprinting of H5N1 avian influenzausing convalescent sera and monoclonal antibodies reveals potentialvaccine and diagnostic targets. PLoS Med, 2009. 6(4): p. e1000049;Krause et al, A broadly neutralizing human monoclonal antibody thatrecognizes a conserved, novel epitope on the globular head of influenzaH1N1 virus hemagglutinin. J Virol, 2011. pmid_21849447; Whittle et al.Broadly neutralizing human antibody that recognizes the receptorbindingpocket of influenza virus hemagglutinin. Proc Natl Acad Sci USA, 2011.108(34): p. 1421621; Yoshida et al., Crossprotective potential of anovel monoclonal antibody directed against antigenic site B of thehemagglutinin of influenza A viruses. PLoS Pathog, 2009. 5(3): p.e1000350).

The inventor hypothesized that reducing the immunogenicity of H5 HAprimary antigenic determinants will increase immune responses againstconserved HA epitopes that do not efficiently elicit immunologicalmemory antibodies under routine infection and seasonal flu vaccineimmunization conditions. These conserved epitopes are present in a broadspectrum of influenzas, and more likely to be retained in future H5N1viruses than are the rapidly evolving primary antigenic determinantstargeted by conventional vaccines, and therefore should representsuperior targets for generating broad H5 and heterosubtypiccross-protection across all HAs.

B-cell epitope characteristics The H5 HA primary antigenic determinantsto be neutralized in this work are defined based on extensive escapemutant and genetic drift data. However, in order to successfully knockthem out and avoid their undesirable replacement with novel antigenicdeterminants, it is necessary to consider the properties of B-cellepitopes. Generally, B-cell epitopes locate to hydrophilic anddynamically flexible sites on a protein's surface (reviewed in HasteAndersen, P., M. Nielsen, and O. Lund, Prediction of residues indiscontinuous B-cell epitopes using protein 3D structures. Protein Sci,2006. 15(11): p. 2558-67., Parker, J. M., D. Guo, and R. S. Hodges, Newhydrophilicity scale derived from high-performance liquid chromatographypeptide retention data: correlation of predicted surface residues withantigenicity and X-ray-derived accessible sites. Biochemistry, 1986.25(19): p. 5425-32., Ponomarenko, J. V. and P. E. Bourne,Antibody-protein interactions: benchmark datasets and prediction toolsevaluation. BMC Struct Biol, 2007. 7: p. 64.; Jin L, Fendly B M, Wells JA, High resolution functional analysis of antibody-antigen interactions.J Mol. Biol. 1992 Aug. 5; 226(3):851-65.). The recent exponential growthof antigen-antibody complex structures in the Protein Data Base enablesdetailed analysis of antigen-antibody contact sites and provides newinformation about the properties of surface substructures that formepitopes on protein antigens. Table 1 is an epitope log-odds ratiopropensity scale (LODrps) derived by analyzing the distribution of aminoacids present on the antigen side of antigen-antibody interfaces in 76different x-ray structures of antigen-antibody complexes (HasteAndersen, P., M. Nielsen, and O. Lund, Prediction of residues indiscontinuous B-cell epitopes using protein 3D structures. Protein Sci,2006. 15(11): p. 2558-67.).

TABLE I epitope log odds ratios for 20 amino acids N 1.242 Y 0.03 R 1.18W −0.064 P 1.164 S −0.145 K 1.136 T −0.233 H 1.098 I −0.713 Q 1.082 F−1.147 D 0.691 V −1.474 E 0.346 A −1.522 M 0.273 L −1.836 G 0.189 C−3.519

A high Discotope log-odds value means that the amino acid isover-represented in the set of structurally well-defined epitopes,whereas under-represented amino acids have low log-odds ratio values.The epitope log-odds ratio propensity scale is particularly useful whenconsidered in conjunction with the extensive evolutionary drift andescape mutant mapping data available for influenza hemagglutinins. Ofthe ten H5 hemagglutinin residues identified as primary antigenicdeterminants on the basis of drift and escape mutant evidence, i.e.,empirically, seven had positive log-odds ratios.

The invention utilizes the epitope log-odds ratio propensity scale inthe design of x-HA mutants with neutralized primary antigenicdeterminants. The strategy for reducing H5 HA primary epitopeimmunogenicity is to replace primary antigenic determinant residues forwhich there is strong drift and escape mutant evidence with amino acidsthat have lower log-odds ratios. This will not only destroy primaryepitope(s), but should also reduce the likelihood of the new surface(s)serving as antigenic determinants. Without being bound by theory, it isbelieved that by disfavoring clonal selection of B cells for primaryantigenic determinants, the immune response should be shifted tosecondary epitopes, including conserved epitopes which do not normallyelicit immune responses due to the immunodominance of HA primaryantigenic determinants.

The invention is further understood by reference to the followingexamples, which are representative and not limiting.

EXAMPLES Example 1 Hemagglutinin Expression and Purification

Recombinant influenza hemagglutinins produced by two differentbaculovirus-based insect cell expression strategies have beendemonstrated to be suitable for vaccine trials (Treanor, J. J., et al.,Dose-related safety and immunogenicity of a trivalentbaculovirus-expressed influenza-virus hemagglutinin vaccine in elderlyadults. J Infect Dis, 2006. 193(9): p. 1223-8.) and for high-resolutionreceptor binding and structural studies (Stevens, J., et al., Structureand receptor specificity of the hemagglutinin from an H5N1 influenzavirus. Science, 2006. 312: p. 404-10., Stevens, J., et al., Structure ofthe uncleaved human H1 hemagglutinin from the extinct 1918 influenzavirus. Science, 2004. 303: p. 1866-70.). Accordingly, baculovirusexpression systems are appropriate for the expression of HA antigens.

The constructs of Stevens contain C-terminal foldon sequences to mediatecorrect folding of the HA, leading to crystallographically verifiedtrimeric HAs, an improvement over the original baculovirus-expressedProtein Sciences HAs of the Treanor reference. Accordingly, the DESexpression approach used, below, is based on the Stevens constructs thatcontain the foldon domain.

A different insect cell expression system, the Drosophila ExpressionSystem (DES) (InVitrogen) was used for production of controlhemagglutinins and xHAs described in the Examples. The DES parentconstruct for parental H5N1 A/Vietnam/1203/2004 (V1203) hemagglutininproduction is illustrated in FIG. 2. The sequence of the expressedrecombinant hemagglutinin is SEQ ID NO: 1 of FIG. 4. The DNA sequenceencoding this protein was inserted into pMT/BiP/V5-His vector, obtainedfrom Invitrogen Corp. The V1203 HA0 precursor sequence was placeddownstream of a metallothionein promoter for inducible expression, and aBIP signal peptide to mediate secretion. A thrombin cleavage site and30-residue foldon and hexahistidine (SEQ ID NO: 12) sequences wereplaced C-terminal to the hemagglutinin-encoding sequence to facilitateHA trimerization, purification and the subsequent removal of the foldonand his tag. The site of HA0 cleavage into HA1 and HA2 was modified(PQRERRRKKRGLFG (SEQ ID NO: 14) to PQRETRGLFG (SEQ ID NO: 15)) in orderto maintain the prefusion conformation, reduce HA cleavage, and promotethe production of HA0 oligomers and trimers, which exhibit superiorimmunogenicity (Wei, C. J., et al., Comparative efficacy of neutralizingantibodies elicited by recombinant hemagglutinin proteins from avianH5N1 influenza virus. J Virol, 2008. 82(13): p. 6200-8.). The HA1 regionin which the primary antigenic determinants are located is shown in amagnification, with the positions of the eleven primary antigenicdeterminant residues targeted by our study marked by black X marks.These primary antigenic determinant residues were selected on the basisof escape mutant data, genetic drift data, and Discotope high log oddsvalues, as explained in Example 2, Table 2.

The HA0 DNA sequence of the construct was optimized for expression inthe DES Drosophila expression system by employing frequently utilizedcodons from a data base of highly expressed Drosophila proteins(Shields, D. C., et al., “Silent” sites in Drosophila genes are notneutral: evidence of selection among synonymous codons. Mol Biol Evol,1988. 5(6): p. 704-16.). The DNA sequence was further modified toinclude translationally silent, unique restriction sites at thelocations shown in the map. Restriction sites in the magnified primaryantigenic determinant region were utilized for rapid, efficient andeconomic substitution of cassettes with changes for x-HA variants.

Expression and Purification of V1203 Hemagglutinin and Related HAs

DES-expressed control hemagglutinins and xHAs were purified using thestrategy described by Stevens and Wilson (Stevens, J., et al., Structureand receptor specificity of the hemagglutinin from an H5N1 influenzavirus. Science, 2006. 312: p. 404-10., Stevens, J., et al., Structure ofthe uncleaved human H1 hemagglutinin from the extinct 1918 influenzavirus. Science, 2004. 303: p. 1866-70.). This protocol produces HAs thatare properly folded with polymerization into trimers resembling thoseobtained from bromelain-released virus hemagglutinin. Thus, such HAsmimic naturally occurring HAs.

The expression system used in the present examples features a thrombincleavage site C-terminal to the HA0 sequence, followed by a “foldon”sequence to promote efficient assembly of the trimer (Frank, S., et al.,Stabilization of short collagen-like triple helices by proteinengineering. J Mol. Biol., 2001. 308: p. 1081-9.), and finally a hexaHis-tag (SEQ ID NO: 12) to facilitate protein purification. Expressionplasmids were DNA sequence verified for the region encoding the signalsequence cleavage site through the His-tag and stop codon, thenco-transfected with pCoHygro (InVitrogen) or pCoPuro (Iwaki, T., et al.,Rapid selection of Drosophila S2 cells with the puromycin resistancegene. Biotechniques, 2003. 35(3): p. 482-4, 486) selection plasmids intoDrosophila S2 cells. Stably transformed hemagglutinin-expressing celllines were selected by growth in the presence of hygromycin or puromycinfor several weeks.

Stably transfected, hemagglutinin-expressing S2 cell lines were expandedup to 500 ml in 10% serum-containing Express Five media (InVitrogen).Then cultures were adapted to serum-depleted conditions by 1:1 dilutionwith serum-free media up to 2 liters HA expression from themetallothionein promoter was induced by addition of CuSO₄, andconditioned medium harvested 3-4 days later at trypan blue cellviabilities of 80-90%. Conditioned media supernatants were prepared, andfrozen at −20 C for cryoprecipitation of insect ferritin and storage.The xHAs were purified by Ni-NTA (Qiagen) chromatography with imidazolegradient elution. Peak fractions, identified by SDS-PAGE, were pooled,buffer exchanged and concentrated into 50 mM NaCl, 10 mM Tris pH 8.Protein concentration was measured by Coomassie Blue dye binding(Biorad). The xHAs used in this study were not thrombin digested, andretained the C-terminal foldon/his-tag sequence to promote recovery oftrimeric and oligomeric hemagglutinins, which are efficient immunogens(Wei et al, J. Virol., 2008, 82:6200). Yields of ˜0.5 mg or more ofhemagglutinin per liter of induced cells were obtained. Reducing andnon-reducing SDS-PAGE was performed to assess purity and confirm HA0status, and the content of monomeric, trimeric and multimeric xHAsassessed by size exclusion chromatography on Sephacryl-S300 HR.

The above DES expression and purification protocols supported efficientproduction of 70 kD HA0 parental V1203 HA and xHAs, which are assembledinto trimeric hemagglutinins. FIG. 3 panel a and b show reduced andnon-reduced SDS-PAGE gels, demonstrating the high level of purity of thepurified xHA antigens, and the absence of inter-HA0 disulfide bonding,which would be indicative of misfolding. Further evidence for the properfolding of the recombinant xHAs was provided by patterns of binding to(1) the 1F02 mAb to the fusion-peptide containing epitope on the HA stem(Wrammert et al., J Exp Med, 2011. 208:181) and (2) the VN04 panel ofmapped mAb antibodies to V1203 HA primary antigenic determinantcontinuous and discontinuous epitopes (Kaverin et al., J Virol, 2007,81:12911). (1F02 and VN04 mAb binding data is presented below in Example2, FIG. 5.) The purified xHAs migrated on S300 Sephacryl gel exclusionchromatography as ˜200 kD trimers and higher MW multimers, as previouslydescribed for baculovirus-expressed HAs (Stevens et al., Science, 2004,303:1866-70; Wei et al., J. Virol., 2008, 82:6200).

Production of xHAs antigens in Drosophila S2 cells is believed toresults in their N-glycosylation with paucimannose and related glycans(Kim Y K, et al., Production and N-glycan analysis of secreted humanerythropoietin glycoprotein in stably transfected Drosophila S2 cells.Biotechnol Bioeng. 2005 Nov. 20; 92(4):452-61). Such glycosylation maybe advantageous for efficient presentation of these molecules to theimmune system via macrophage, monocyte and dendritic cell mannosereceptors (Buzás E I et al., Carbohydrate recognition systems inautoimmunity. Autoimmunity. 2006 December; 39(8):691-704; Gazi, U. andL. Martinez-Pomares, Influence of the mannose receptor in host immuneresponses. Immunobiology, 2009. 214(7): p. 554-61). FIGS. 2c and 2d GNAlectin blotting and PNGase F digestion experiments confirmed thepresence of mannose terminated Nglycans on the xHAs.

These results demonstrate successful Drosophila expression systemsynthesis and purification of recombinant hemagglutinins for inductionof antisera and evaluation of the immune responses.

Example 2 Design and Production of xHA Mutants

V1203 primary antigenic determinants were ablated in x-HA variants bysubstitutions at epitope residues identified on the basis of escapemutant and genetic drift data. These positions were replaced with aminoacids that are present at low frequencies on the antigen side of contactsites in a database of antigen-antibody crystal structures. Knockout ofV1203 specific primary antigenic determinants in xHA variants wasverified by screening against a panel of well-characterized monoclonalantibodies (Kaverin et al., J Virol, 2007, 81:12911) obtained from theBiodefense and Emerging Infections Research (BEIR) repository.

Mapping of the Primary Antigenic Determinants

The first step in the design of the xHAs was to carefully analyze H5N1antigenic drift and escape mutant data and to develop operationaldefinitions of the primary antigenic determinants that will be knockedout. Table 2 presents the H5 A/Viet/1203/04 HA1 ectodomain sequence fromresidues 125-209 (H3 numbering), with primary antigenic determinantresidues shaded. Black indicates primary antigenic determinant residuesfor which differences from the A/Viet/1203/04 sequence have beenobserved both in laboratory-generated escape mutants and in naturaldrift isolates (human viruses, 1997-2004). Gray indicates residues forwhich the assignment as a primary antigenic determinant was made solelyon the basis of escape mutant evidence. Light gray indicates residuesfor which there is only evolutionary drift evidence.

TABLE 2 MAPPING OF H5 HEMAGGLUTININ PRIMARY ANTIGENIC DETERMINANTS(Table discloses residues 138-225 of SEQ ID NO: 1)

Footnotes of Table 2 ¹ H5 A/Viet/1203/04 HA residue ID; H3 numbering ² H= Primary antigenic determinant assignment based on drift in 1997-2005H5N1 human isolates, Stevens, 2006, FIG. S5 (Stevens, J., O. Blixt, etal. (2006). “Structure and receptor specificity of the hemagglutininfrom an H5N1 influenza virus.” Science 312(5772): 404-10.) ³ A = Primaryantigenic determinant assignment based on drift in 2003-2006 H5N1 humanand avian isolates, Kaverin, 2007, Table 4 (Kaverin, N. V., I. A.Rudneva, et al. (2007). “Epitope mapping of the hemagglutinin moleculeof a highly pathogenic H5N1 influenza virus by using monoclonalantibodies.” J Virol 81(23): 12911-7.) ⁴ M = Primary antigenicdeterminant assignment based on escape mutants of H5N2A/Mallard/Pennsylvania/10218/84 from various mAbs, Kaverin, 2002, Tables3 (Kaverin, N., et al. (2002). “Structure of antigenic sites on thehaemagglutinin molecule of H5 avian influenza virus and phenotypicvariation of escape mutants.” J Gen Virol. 83: 2497-505.) ⁵ x = Primaryantigenic determinant assignment based on escape mutants of H5A/Vietnam/1203/04 from indicated monoclonal antibody, Kaverin, 2007,Table 1 and 2 X = Primary antigenic determinant assignment based on H5N2A/Mallard/ Pennsylvania/10218/84 escape mutants (Kaverin, 2002) with theindicated mAb. ⁶ D = predicted using DiscoTope algorithm for theidentification of discontinuous B-cell epitopes, Haste Andersen, 2006www_cbs_dtu_dk/services/DiscoTope *Asterisks mark amino acids withepitope log odds ratio values of >0.3 (Haste Andersen, P., M. Nielsen,et al. (2006). “Prediction of residues in discontinuous B-cell epitopesusing protein 3D structures.” Protein Sci 15(11): 2558-67.) Shadingshows primary antigenic determinant residues. Assignments were based ondrift and escape mutant evidence (black), drift-only evidence (lightgray), or escape mutant only data (gray) The baculovirus expressed H5A/Viet/1203/04 HA of Stevens (2006) is N-glycosylated on Asn-169 but notAsn-158.

Ablation of Primary Antigenic Determinants

Discotope log odds ratio propensity scale (LODrps) values for the 20amino acids have been assigned based their relative frequencies on theantigen side of the antigen-antibody interfaces in 76 different x-raystructures (Haste Andersen, P., M. Nielsen, and O. Lund, Prediction ofresidues in discontinuous B-cell epitopes using protein 3D structures.Protein Sci, 2006. 15(11): p. 2558-67). A high LODrps means that theamino acid is over-represented in known, structurally well-definedepitopes, whereas under-represented amino acids have low LODrps values.

In the above Table 2 presentation of H5 HA primary antigenic determinantsequences, residues with high LODrps values are marked with asterisks(*). Of the 34 shaded primary antigenic determinant residues in Table 2,11 are surface-exposed and have log-odds ratio values of >0.3. Theinventor hypothesizes that replacing these residues with low or negativelog-odds ratio amino acids (see Table 3) should not only knock outprimary antigenic determinants, but also reduce the likelihood of themodified surfaces also serving as new antigenic determinants.

TABLE 3 DESIGN PLAN FOR PRIMARY ANTIGENIC DETERMINANT KNOCK-OUT INCONTROL AND x-HEMAGGLUTINIS

¹ Escape mutant substitutions are shown in bold face forA/Vietnam/1203/04 (H5N1) escape mutants, and in italics for forA/Mallard/Pennsylvania/10218/84 (H5N2) escape mutants ² H3 numbering isused throughout the Table. ³ Log odds ratio propensity score (LODrps)values in parentheses. ⁴ Wildtype H5 A/Viet/1203/04 residues are shownin white cells, and substitution mutations are in gray cells. ⁵ LODrpsindex was calculated from sum of Discotope LODrps scores of HA primaryantigenic determinant residues. Higher values indicate that primaryantigenic determinants are richer in amino acids that occur commonly onthe anitigen side of antibody-epitope contact sites. Lower valuesindicate that primary antigenic determinants are composed of amino acidswhich are under represented on the antigen side of antibody-epitopecontact sites.

x-HA Design

Table 3 summarizes the design of H5 x-HA antigens. The parent moleculewas HA0 from A/Viet/1203/04 (clade 1), a highly pathogenic H5N1influenza isolated from a Vietnamese patient in 2004. A/Indonesia/5/05(clade 2.1.3) and A/Egypt/2782/06 (clade 2.2) hemagglutinins serve asdrift controls.

For a given macromolecular antigen, and especially for influenzahemagglutinins, some substructures stimulate immune responses veryeffectively, while other substructures do not. The common properties ofprotein substructures that are effective B-cell antigens include surfacelocation, hydrophilicity and dynamic flexibility (Haste Andersen, P., etal., Prediction of residues in discontinuous B-cell epitopes usingprotein 3D structures. Protein Sci, 2006. 15(11): p. 2558-67., Parker,J. M., D. Guo, and R. S. Hodges, New hydrophilicity scale derived fromhigh-performance liquid chromatography peptide retention data:correlation of predicted surface residues with antigenicity andX-ray-derived accessible sites. Biochemistry, 1986. 25(19): p. 5425-32.,Ponomarenko, J. V. and P. E. Bourne, Antibody-protein interactions:benchmark datasets and prediction tools evaluation. BMC Struct Biol,2007. 7: p. 64). Certain amino acids occur more frequently in epitopescompared to others (Jin L et al., J Mol. Biol. 1992, 226(3):851), andthis is reflected in their Discotope LODrps values as illustrated inTable 1 (Haste Andersen, P, Protein Sci, 2006. 15(11): p. 2558-67).

Accordingly, x-HAs were generated by altering the steric shapes ofprimary antigenic determinants towards reducing their immunogenicity.Replacement amino acids are low or negative LODrps residues that occurrarely in stable antigen-antibody interfaces. Primary antigenicdeterminant ablation is performed by replacing primary antigenicdeterminant residues identified by the drift and escape mutant data ofTable 2 with amino acids that have lower LODrps values according to thescheme detailed in Table 3. Reducing the tendency of antibodies to bindprimary antigenic determinant sites by replacement of positive LODrpsamino acids with low or negative LODrps amino acids should preventclonal expansion of B-cells producing antibodies to the original primaryantigenic determinants as well as the “knocked out” sites, and therebyfacilitate expansion of B-cells making antibodies to conserved, normally“immunorecessive” sites on the hemagglutinin molecule that do notroutinely elicit detectable immune responses.

During the design process, it is important to avoid changes that induceprotein-folding problems in regions distal to the primary antigenicdeterminants, because these regions may contain structures that willbecome “secondary” epitopes. Therefore, modifications were limited tothe confines of well-defined primary antigenic determinants. The x-HA.4and xHA.5 variants, have all 5 primary antigenic determinants knockedout, but by different combinations of amino acid substitutions (rightside of Table 3). In theory, ablation of all 5 epitopes should directthe immune response to conserved epitopes. In contrast, the x-HA.1,x-HA.2, and x-HA.3 hemagglutinins were designed to eliminate, as well asto retain, different subsets of primary antigenic determinants. Theseserve as experimental controls, but may also elicit immune responses toconserved epitopes.

For the x-HA.4 series, 6 high LODrps residues in the V1203 primaryantigenic determinants were replaced with escape mutant residues inx-HA.4a, or mainly threonine (LODrps=−0.233) in x-HA.4b, or mainlyalanine (LODrps=−1.522) in x-HA.4c. Based on detailed analysis of the2FK0 trimer structure, escape mutant and drift data, modification ofjust 6 residues should completely ablate the HA primary antigenicdeterminants. x-HA.5 series hemagglutinins, with 10 changes in primaryantigenic determinant residues should further eliminate residualantigenicity in the primary epitopes.

Note that 2 residues, which are understood to contribute to the primaryantigenic determinants based on escape mutant data, were not altered.These are I155, which already has a low LODrps value (−0.713), and D187,which is at the edge of the receptor binding domain (RBD). This decisionwas based on a D187 substitution perturbing the nearby RBD, and therebydisrupting sialic acid binding. The other reason for keeping theconserved D187 residue is that, by preserving the RBS, it is possible togenerate an immune response against the RBS. Such an antibody would bean ideal neutralizing antibody as has been recently demonstrated(Whittle, J. R., R. Zhang, S. Khurana, L. R. King, J. Manischewitz, H.Golding, P. R. Dormitzer, B. F. Haynes, E. B. Walter, M. A. Moody, T. B.Kepler, H. X. Liao, and S. C. Harrison, Broadly neutralizing humanantibody that recognizes the receptor binding pocket of influenza virushemagglutinin. Proc Natl Acad Sci USA, 2011. 108(34): p. 14216-21).

In the bottom row of Table 3 a measurement called the LODrps index wasintroduced. It is calculated by summing the LODrps values for primaryantigenic determinant residues of the HA in each column and serves as arelative indicator of the degree to which the primary antigenicdeterminants are neutralized in a given hemagglutinin molecule. Theparental V1203 HA control has a LODrps index value of 6.7, which issimilar to the 4.6 and 6.3 values obtained for the other naturallyoccurring Indo05 and Egypt06 control hemagglutinins. Partialneutralization of V1203 primary antigenic sites one mAb epitope at atime in x-HA.1, x-HA.2 and x-HA.3, modestly reduced the 6.7 index valueto around 4. However, (almost) wholesale replacement of V1203 primaryantigenic determinant residues with escape mutant substitutions inx-HA.em reduced the index value to 0.6. For the x-HA.4 series in which 6primary epitope residues were substituted with escape mutant residues,threonine, or alanine, index values were 0.4, −3.2 and −9.6,respectively. For the x-HA.5 series, the index values were −9.2 and−15.6. The inventor hypothesizes that, by reducing the LODrps indices ofx-HA mutants, modified surfaces of the HA becomes less antigenic, andthereby to switch the immune response to remaining higher LODrpsresidues of conserved HA surfaces that are not normally immunogenic.

Immunoassays Verify Proper Folding of Recombinant Hemagglutinins andPrimary Antigenic Determinant Knock Out in xHAs

Recombinant xHA protein folding was assessed by testing for binding towell-characterized mAbs which recognize discontinuous epitopes on the HAhead and stem. Human mAb 1F02 recognizes the conserved fusionpeptide-containing epitope on Group 1 HA stems and protects mice from invivo virus challenge with antigenically distinct influenzas (Wrammert etal. J Exp Med, 2011. 208(1): p. 181-193). The VN04 mAbs recognize 3discontinous epitopes, and 1 linear epitope, within the primaryantigenic determinant surfaces of the V1203 HA (Kaverin et al., J Virol,2007, 81:12911). Binding was assayed by immobilizing xHAs, or a controlprotein (human antithrombin III), at 1 ug/ml on microplates, followed byblocking and serial exposure to 1F02 and alkaline phosphatase-goat antihuman Ig, or to the VN04 mAbs and alkaline phosphatase-goat anti mouseIg. FIG. 5 shows that the 1F02 mAb to the HA stem epitope bound all ofthe xHAs, whereas the VN04 mAbs to V1203 primary antigenic determinants,bound to xHA.par (which has a wildtype HA head surface with intactprimary antigenic determinants), but not xHA.4b and 5a (which haveablated immunodominant epitopes, see Table 3). Together, the bindingdata are supportive of proper folding of the xHA.par and the xHA.4b and5a hemagglutinins, and also confirm successful knock-out of primaryantigenic determinants in the latter.

Example 3 Production of Antisera to H5 Control and x-HA Hemagglutinins,Surrogate Assay for Viral Neutralization Function, and Identification ofx-HA Universal Vaccine Candidates

Overview

The abilities of control and x-HA hemagglutinins to elicit broadlycross-protective humoral antibody responses were examined in mice.Antisera raised to individual control and x-HA hemagglutinins, as wellas antisera raised by sequential challenge with different x-HAs, werescreened in HA pseudotyped lentiviral vector reporter neutralizationassays to identify molecules that stimulate responses to stable,conserved regions of H5 hemagglutinins.

Production of Mouse Antisera to Recombinant Hemagglutinins

Non-immune serum is collected from mice. Groups of 4 or 5 Balb/C or fVBmice are vaccinated by injection with 10 or 20 ug of a control or x-HAhemagglutinin in Sigma Adjuvant System (S6322, formerly Ribi AdjuvantSystem). Three weeks after the primary injection and at >21 dayintervals after each boost, animals received additional 10 ug boosts.Blood was drawn 1-2 weeks after the booster immunizations. Mice areimmunized with a single control or x-HA hemagglutinin repeatedly, orreceive different x-HA antigens for the original immunization andsubsequent boosts an alternative immunization protocol.

Characterization of x-Ha Cross-Protective Function Using InfluenzaHemagglutinin Pseudotyped Lentiviral Vector Reporter Assays

If an x-HA is to serve as an effective avian influenza pandemic vaccineand antigen for immunotherapeutic development, it must elicit immuneresponses to hemagglutinins from a wide range of H5 influenzasindependently of sharing primary antigenic determinants with evolvingstrains, and those immune responses must lead to the neutralization ofviral function in order to achieve cross protection. A lentiviral vectorreporter assay is used to quantify the ability of antisera raisedagainst various control and x-HA hemagglutinins to inhibit HA-mediatedmembrane fusion, which is a essential step in the infection process andviral life cycle.

The HA pseudo virus lentiviral vector reporter assay was developed, andthe components were generously provided, by Dr. Gary Nabel andcolleagues from the NIH Vaccine Research Center (Kong, W. P., et al.,Protective immunity to lethal challenge of the 1918 pandemic influenzavirus by vaccination. Proc Natl Acad Sci USA, 2006. 103(43): p.15987-91.). To generate the flu HA pseudotyped reporter viruses, 293Tcells were cotransfected with 7 ug of pCMVΔR8.2, 7 ug of pHR′CMV-Luc,and 125 ng of a CMV/R 8κB H5 construct, wherein the HA segmentcorresponds to H5 A/Vietnam 1203/04 (clade 1), A/Thai1(KAN-1)/04 (clade1), A/Indonesia/5/05 (clade 2.1.3), A/Egypt/2782/06 (clade 2.2),A/Nigeria/641/06 (clade 2.2), or A/Iraq/207/06 (clade 2.2). Thepackaging cells are transfected overnight, then changed to fresh medium.At 48 h, virion-containing supernatants were harvested, 0.45-umfiltered, aliquoted, and used immediately, or frozen at −80° C.

For neutralization assays, antiserum dilutions are mixed withlentiviruses that have been pseudotyped with the different H5hemagglutinins, then added to 96-well plates containing 5,000 293A cellsper well. The medium is changed 14-16 h later, and at 72 or 96 h postinfection, cells are lysed and the lysates assayed for luciferaseactivity (Promega Bright Glo assay). Percent neutralization of the LVreporters by tested antisera is calculated as {1−[(luminescence in wellswith added antiserum)/(luminescence in wells with no added antiserum)]}.Lentivirus neutralization titers are obtained by analyzingneutralization as a function of antiserum dilution. For example, LVnt50is the greatest antiserum dilution producing at least 50% inhibition ofneutralization. The HA-pseudotyped LV reporter assay is used to screenantisera and identify xHAs inducing neutralizing antibodies to conservedHA features. The desired xHAs will elicit antisera with high titers forthe neutralization of multiple HA pseudotypes. For the x-HA.4 and x-HA.5series in which all primary antigenic determinants are knocked out andreplaced in 4 out of 5 cases with very low LODrps amino acids, wepredict that the immune response will be switched to substructures onthe HA surface that are not normally immunogenic, and that if suchsubstructures are conserved, the antisera will bind and neutralizereporter viruses pseudotyped for all the clades.

Targets of Antibodies to x-HA s with Universal Vaccine Function

Epitope identification algorithms such as DiscoTope (Haste Andersen, P.,M. Nielsen, and O. Lund, Prediction of residues in discontinuous B-cellepitopes using protein 3D structures. Protein Sci, 2006. 15(11): p.2558-67) are used to predict the locations of the secondary antigenicdeterminants. On the right side of Table 2, residues of theA/Viet/1203/04 HA ectodomain that DiscoTope predicts to be epitopes aremarked. The Discotope algorithm predicted 34 residues as epitopes. Onthe basis of genetic drift and escape mutant data, 12 of the 34correspond to HA primary antigenic determinants. The remaining 18residues, not mapped to known primary antigenic determinants,potentially include some conserved secondary antigenic determinants.

Conserved hemagglutinin structural features mediating viral functionswhose interruption causes neutralization include: (1) sites in andbordering the receptor binding site, and (2) stem fusion peptidestructural elements participating in the conformational change thatmediates viral envelope fusion with the host cell membrane (Skehel, J.J. and D. C. Wiley, Receptor binding and membrane fusion in virus entry:the influenza hemagglutinin. Annu Rev Biochem, 2000. 69: p. 531-69.).

The inventor hypothesizes that (1) structural neutralization of HAprimary antigenic determinants shifts the immune response towardsconserved epitopes, and that (2) sequential immunization with differentx-HAs (Table 5, lines 13 and 14) will be superior for eliciting broadlyprotective immune responses.

Example 4 Cross Neutralization Results

Table 4 shows experiments investigating neutralization of clade 1, clade2.2 and clade 2.1.3 H5 HA-pseudotyped lentiviruses by antisera raisedagainst recombinant xHA immunogens bearing intact wildtype (xHA.par),and partially (xHA.2) and completely (xHA.4b and xHA.5a) ablated primaryantigenic determinants. Mouse non-immune sera did not neutralize any ofthe reporter viruses. Antisera generated with the V1203 xHA.par parentalcontrol exhibited a focused pattern of neutralization, which was limitedto the V1203 clade 1 and Egypt clade 2.2 pseudotypes. Broader crossneutralization, extending to the H5 Indonesia clade 2.1 and H1 PR8reporters, was obtained with antisera to xHA.2, which has partialablation of hemagglutinin primary antigenic determinants. Additionally,antisera raised by priming with xHA.4b and subsequent xHA.5a boostingalso produced broad cross neutralization. For reference, theneutralization pattern obtained with purified mAb 1F02 (Wrammert et al.J Exp Med, 2011. 208(1): p. 181-193) is shown in the Controls section atthe bottom of the Table 4. The 1F02 mAb binds a conserved, fusionpeptide-containing epitope on the HA stem and provides in vivoprotection against several antigenically distinct influenzas whenadministered therapeutically. The broad in vitro neutralization observedwith the polyclonal antisera from xHA immunized animals, may result fromcontributions of antibodies like 1F02, which recognize conservedelements on the HA stem, and/or from recently described antibodies toconserved elements on the HA head, which interfere with receptorbinding.

The broadened neutralization results were obtained for a portion of themice immunized with xHAs, indicating that that ablation of primaryepitopes can promote an immune response with potential to protectagainst different serovariants. Different adjuvant strategies may beapplied to increase the reproducibility and magnitude of the response.

TABLE 4 BROADENED NEUTRALIZATION OF INFLUENZA HEMAGGLUTININ PSEUDOTYPEDLENTIVIRUS REPORTERS BY xHA ANTISERA LVNT50¹ LVNT50 LVNT50 LVNT40² mouseserum V1203 Indo Egypt PR8 ID from³ H5 clade 1 H5 clade 2.1 H5 clade 2.2H1 non-immune serum; 2 BALB/c mice bD.LR n.a ∘ ∘ ∘ ∘ bE.LR n.a. ∘ ∘ ∘ ∘xHA.par: 10 ug prime and boosts; 2 BALB/c mice bA.L b2 >912 ∘ >3648 ∘bA.R b3 >1706 ∘ >6823 ∘ xHA.2: 20 ug prime and 10 ug boosts; best 2 of 3BALB/c mice A4.0 b6 >5472 ∘ >2736 >342 A4.L b6 >685 ∘ >2742 >1371 xHA.2:10 ug prime and boosts; best 2 of 5 BALB/c mice bB.LR b4 >3655∘ >3655 >114 bB.R b4 >914 >914 >3655 ∘ 20 ug xHA.4b prime and 10 ugxHA.5a boosts; best 3 of 4 BALB/c mice B2.0 b7 >914 >228 >3655 >457B2.LR b4 ∘ ∘ >2742 >343 B2.R b5 ∘ ∘ >457 >457 10 ug xHA.4b prime and 10ug xHA.5a boosts; best 2 of 5 BALB/c mice bC.LR b4 ∘ >171 >2742 ∘ bC.Rb5 >533 >133 >4265 >133 10 ug xHA.4b prime and 10 ug xHA.5a boosts; best2 of 4 fVB mice fC.L b3 >914 >914 >3655 ∘ fC.LL b5 >685 >685 >5483 >685CONTROLS mAb 1F02, >228 ∘ >1824 >114 32 ug/ml rabbit α H5N1 >22800∘ >11400 ∘ rgA/Viet/1203/04 (BEIR NR-4485) goat α H5 HAA/ >91200∘ >18240 ∘ tern/South Africa/61 (BEIR NR-3156) goat α H1 HAA/ ∘ ∘∘ >54825 Puerto Rico/8/1934 (BEIR NR-3148) ¹LVnt50, serum dilutionproducing >50% neutalization ²LVnt40, serum dilution producing >40%neutalization ³b2, boost 2; b3, boost 3; n.a., not applicable

Example 5 xHAs Elicit Antibodies to Conserved Fusion-Peptide ContainingEpitopes on the HA Stem

When administered therapeutically, monoclonal antibodies to conservedfusion peptide-containing epitopes on the hemagglutinin stem mediatebroad influenza protection. Therefore, it is of interest to determine ifxHAs elicit immune responses to the conserved stem epitopes and could beused to strengthen immunological memory for and induce production ofbroadly protective stem antibodies. Example 5 and FIG. 6 show that mouseimmunization with xHAs elicits antibodies to this critical HA stemelement.

Monoclonal antibody 1F02 binds to a conserved fusion peptide-containingepitope on Group 1 HA stems and protects mice from live virus challengeby antigenically distinct influenzas (Wrammert et al. J Exp Med, 2011.208(1): p. 181-193). mAb 1F02 was used in a competition elisa todetermine if antibodies to conserved fusion peptide-containing epitopeson the HA stem are present in polyclonal antisera from mice immunizedwith xHAs. The solid phase for the assay was BEIR NR-4143rgA/Vietnam/1203/04 (H5N1) monovalent influenza sub-virion vaccine, withits hemagglutinin element derived from V1203. NR-4143-coated wells wereexposed to dilutions of anti-xHA mouse antisera, or to non-immune serum,or control mouse mAb C179 (Okuno, Y., Y. Isegawa, F. Sasao, and S. Ueda,A common neutralizing epitope conserved between the hemagglutinins ofinfluenza A virus H1 and H2 strains. J Virol, 1993. 67(5): p. 2552-8) asnegative and positive competition controls, respectively; followed byincubation with 1.2 ug/ml human mAb 1F02. 1F02 binding was measured withalkaline phosphatase goat anti-human Ig. Percent 1F02 binding values,calculated as (A405 in the presence of antiserum)/(A405 in the absenceof antiserum), were used to assess the presence and level of stemfusion-peptide epitope binding antibodies in the serum samples.

Results of 1F02 competition experiments are presented in FIG. 6. Panel Ashows no competition by mouse non-immune serum (m NI, open circles), andcompetitive, concentration-dependent reduction of 1F02 binding followingexposure to the positive control C179 mAb (solid triangles). Each of theplots in the remaining B-D panels presents 1F02 competition results fromserum samples obtained at various stages during the immunization of asingle animal with xHAs. The designation “pr” indicates antiseraobtained after priming, “1” and “b2” are respectively antisera obtainedafter boosts 1 and 2, etc. The animals in panel B were primed andboosted with xHA.par (10 ug/injection). The animals in panels C and D,were primed with xHA.4b, then repeatedly boosted with xHA.5b; those in Creceived 10 ug doses of the xHA antigens, while those in D received 20ug priming and 10 ug boosting doses.

Antisera obtained after priming did not contain measurable 1F02competitive activity. But in the selected animals shown, 1F02-competingantibodies developed subsequently, appeared in sera collected after thefirst, second or third boost, and persisted once they had appeared.Surprisingly, anti-stem antibodies were present not only inxHA.4b-primed and xHA.5a-boosted mice (C and D), but also in animalschallenged solely with wildtype xHA.par (B). Flu infections and seasonalvaccines are thought to elicit only limited production of anti-stemantibodies. However, the HA stem of the recombinant xHA.par immunogen,which is comprised of HA0 trimers and oligomers, may be more accessiblethan are HA stems contained in the virion envelope or vaccinepreparations, which undergo formalin cross-linking during manufacture.Additionally, adjuvants expand and enhance antibody responses toinfluenza (Coler, R. N. et al., A synthetic adjuvant to enhance andexpand immune responses to influenza vaccines. PLoS One, 2010. 5(10): p.e13677; Khurana, S., et al., Vaccines with MF59 adjuvant expand theantibody repertoire to target protective sites of pandemic avian H5N1influenza virus. Sci Transl Med, 2010. 2(15): p. 15ra5.). Accordingly,adjuvanted (Sigma Adjuvant System) xHA.par administration may have beenanother factor in the production of anti-stem antibodies for this group.

Neutralization and 1F02 competition results were not tightly correlated,consistent with a polyclonal immune response to xHA antigens. Theneutralization and 1F02 competition result disparities imply thatantibodies to conserved structures above and beyond the fusionpeptide-containing stem epitope also contribute to neutralization.These, for example, might include broadly neutralizing antibodies whichbind to the HA head and inhibit RBS access.

Example 6 xHAs Elicit Antibodies to HA Head Epitopes

Hemagglutination inhibition (HAI) assays were performed to determine ifxHAs induced antibodies that interfered with red blood cell (RBC) sialicacid binding to the HA receptor binding sites of three different H5 andone H1 hemagglutinin. Antigens were HA-pseudotyped lentiviruses of thespecificity indicated by row labels. LVs were incubated for 30 min withdilutions of receptor destroying enzyme (RDE) treated antisera, or noserum, as indicated by column labels in panels B-D. Plates werephotographed 40 m after turkey RBC addition. RBC buttons in the bottomright corner wells of each panel are RBC-only, no-hemagglutinationcontrols. Clear appearance of the four wells above theno-hemagglutination controls indicates HAI by the respective LVantigens. Comparisons of the three left wells of bottom rows withadjacent no-hemagglutination control show that RDE-treated antisera hadno inherent hemagglutinating activity. Presence of RBC buttons ofvarious sizes in wells where antiserum was incubated with LV antigenindicates inhibition of hemagglutination by antibodies contained in theantiserum, and demonstrates that xHA immunization can induce productionof antibodies that reduce interactions between HA head receptor bindingsites and RBC sialic acids. Panel A shows controls for mouse (M) andrabbit (R, BEIR NR-4484) nonimmune (NI) sera, a polyclonal rabbitantiserum (BEIR NR-4487) to V1203, and a polyclonal goat antiserum(NR-3148) to PR8. Antisera for the panel B experiment were from miceprimed and boosted with xHA.par (10 ug/injection). Antisera for thepanel C experiment were from mice primed with xHA.4b, then boosted 4times with xHA.5b using 10 ug of the xHA antigens. Antisera for thepanel D experiment were from mice primed with xHA.4b, then boosted 6times with xHA.5b (20 ug primes and 10 ug boosts).

Sera from mice immunized with the parental control xHA.par hemagglutininbearing intact primary antigenic determinants produced expectedinhibition of hemagglutination (panel A). Panels C and D show HAIresults from animals primed with xHA.4b and boosted with xHA.5b. Theprimary antigenic determinants on xHA.4b and xHA.5a were ablated withdifferent combinations of low LODrps amino acid substitutions (see Table3 and FIG. 5). Hemagglutination inhibition was also noted for someanti-xHA.4b-5a antisera, indicating production of antibodies tosecondary epitopes that are adjacent to the receptor binding site, orwhich alter the conformation of the RBS when the antibodies are bound.Thus, xHA.4b and xHA.5a, which have different primary antigenicdeterminant surfaces than the HAs on the conventional hemagglutinatingantigens, can elicit antibodies to HA head region sites that aredistinct from the primary antigenic determinants, but neverthelessreduce RBS access. Several antibodies to conserved epitopes on the HAhead mediate broad influenza protection (Khurana et al., PLoS Med, 2009.6(4): p. e1000049; Krause et al., J Virol, 2011. pmid_21849447; Whittle,et al., Proc Natl Acad Sci USA, 2011. 108(34): p. 1421621; Yoshida, etal., PLoS Pathog, 2009. 5(3): p. e1000350). The HA inhibition observedwith xHA.4b-5a induced antibodies observed suggests that thereceptor-binding inhibition observed with those antibodies are importantin contributing to the broad influenza neutralization in animalsimmunized with these xHAs.

Example 7 xHA.5a Primary Antigenic Determinant Ablations Broaden ImmuneResponse to H5 Influenzas

TABLE 5 LVnt50¹ titers from xHA.5a immunized mice MOUSE ANTISERA²HA-pseudotype of LV reporters immunogen serum³ V1203 indo egypt PR8xHA.par b2 >912 ∘ >3648 ∘ xHA.par b3 >1706 ∘ >6823 ∘ xHA.5a b2 ∘ ∘ >3648∘ xHA.5a b2 >228 ∘ >1824 ∘ xHA.5a b2 >1824 >456 >3648 ∘ xHA.5ab2 >912 >228 >3648 ∘ none n.a. ∘ ∘ ∘ ∘ none n.a. ∘ ∘ ∘ ∘ none n.a. ∘ ∘ ∘∘ CONTROLS goat anti H5 HAA/tern/ >91200 ∘ >182400 ∘ South Africa/61c(BEIR NR-3156) rabbit anti H5N1 rgA/ >22800 ∘ >11400 ∘ Viet/1203/04(BEIR NR-4485) mAb 1F02, 56 ug/ml >456 ∘ >3648 >114 ¹LVnt50, serumdilution producing >50% neutalization ²each row is a different mouse³b2, boost 2; b3, boost 3; n.a., not applicable

Table 5 shows that mice challenged with xHA.5a, an H5A/Viet/1203/2004-derived hemagglutinin with knock out of all primaryantigenic determinants (see Table 3 and FIG. 5), produced polyclonalantisera with broader H5 neutralization than did mice challenged withthe parental xHA.par control hemagglutinin, which retains the intact,wild type primary antigenic determinants of V1203. Given that the V1203primary antigenic determinants were not present on the xHA.5a immunogen,it is likely that the broadly neutralizing antibodies it elicited in 2mice recognize H5 structural elements that are well-conserved across theH5 subtype. The significance of Example 7 is that it demonstrates that aresponse can be elicited with only one xHA. This suggests that a broadlyprotective vaccine against circulating and future pandemic H5 avianinfluenzas could be developed from an xHA.5a platform.

The foregoing examples demonstrate that xHAs are properly folded andform the correct prefusion trimeric form, and therefore present as manyof the conserved epitopes as possible, including epitopes that have notbeen identified. These xHAs elicit POLYCLONAL responses containingantibodies to a variety of different conserved HA structures, each ofwhich mediate distinct essential functions in the virus life cycle. Forexample, the combined presence of (a) antibodies to conserved epitopeson the head and (b) antibodies to conserved stem epitopes would blockvirus replication at 2 different points in the life cycle (infection andfusion), which are more effective than blocking either step alone.

The reported xHA approach is fundamentally different from widelyproposed universal flu vaccine development strategies of isolatingbroadly neutralizing mAbs, precisely mapping their epitopes, and usingthis information for structure-based design of immunogens that willelicit antibodies focused around the mapped epitope. Instead, xHAs are‘generic’ hemagglutinin molecules, which have knocked-out immunodominantepitopes, but retain normal overall HA tertiary and quartenarystructure, including multiple conserved elements shared by distantlyrelated influenzas. In contrast to peptide-based and truncated HAuniversal vaccine candidates, trimeric xHAs provided a single immunogento elicit antibodies to multiple conserved epitopes, leading topolyclonal anti-xHA antisera containing antibodies to conserved,neutralization-mediating HA stem and head targets. This finding supportsthe feasibility of developing long-lasting, broadly neutralizing,subunit-type universal flu vaccines from an xHA platform. xHA vaccinesare also suitable for both stimulating and amplifying “seasoned”pan-influenza immunity, wherein cross-protection is mediated by thecombined effects of complementary, broadly neutralizing antibodies. Thisrepresents a promising development because it means that xHA-baseduniversal flu vaccines should be able to work in adults through amechanism of boosting conserved epitope memory cells, rather thanpriming.

What is claimed is:
 1. A method of making a vaccine that elicits animmune response against non-immunodominant conserved epitopes on aninfluenza H5 haemagglutinin, comprising: (a) identifying one or moreprimary antigenic determinant residues of a primary immunodominantepitope of a native influenza H5 haemagglutinin, wherein the primaryimmunodominant epitope comprises amino acids 138-209 of SEQ ID NO:1, andwherein the identification of the primary antigenic determinant residuescomprises; comparing the amino acid sequence of the native influenza H5haemagglutinin to the corresponding amino acid sequence of an escapemutant and/or a genetic drift isolate; and identifying one or more aminoacids that differ between the amino acid sequence of the nativeinfluenza H5 haemagglutinin and the amino acid sequence of the escapemutant and/or the genetic drift isolate; (b) determining the log oddsrelative propensity scale (LODrps) value of one or more of the primaryantigenic determinant residues; (c) modifying at least one of theprimary antigenic determinant residues having a high LODrps value byreplacing the at least one of the primary antigenic determinant residueshaving a high LODrps value with an amino acid having a low LODrps valuethereby creating a modified influenza H5 haemagglutinin with reducedantigenicity, wherein the at least one modified primary antigenicdeterminant residue of amino acids 138-209 of SEQ ID NO:1 is selectedfrom the group consisting of Ser141Ala, Ser141Val, Ser141Leu, Ser141Ile,Ser144Ala, Ser144Val, Ser144Leu, Ser144Ile, Glu146Ser, Glu146Thr,Glu146Val, Glu146Leu, Glu146Ile, Pro156Ala, Pro156Val, Pro156Ile,Gln158Thr, Gln158Val, Lys160Leu, Ser161Val, Ser161Ile, Lys172Ser,Lys172Ala, Lys172Val, Lys172Leu, Thr176Leu, Arg182Ala, Arg182Leu,Lys209Ala, and/or Lys209Val; and (d) utilizing the modified influenza H5haemagglutinin to induce antibodies against the non-immunodominantconserved epitopes.
 2. The method of claim 1, wherein each of theprimary antigenic determinant residues having a high LODrps value isreplaced with an amino acid having a low LODrps value.
 3. A vaccine madeby the method of claim
 1. 4. A modified influenza H5 haemagglutinin inwhich at least one primary antigenic determinant residue of a primaryimmunodominant epitope in a native influenza H5 haemagglutinin ismodified by replacement of at least one high LODrps amino acid with alow LODrps amino acid, thereby reducing the antigenicity of the primaryimmunodominant epitope, wherein the primary immunodominant epitopecomprises amino acids 138-209 of SEQ ID NO:1, and wherein the at leastone modified primary antigenic residue of amino acids 138-209 of SEQ IDNO:1 is selected from the group consisting of Ser141Ala, Ser141Val,Ser141Leu, Ser141Ile, Ser144Ala, Ser144Val, Ser144Leu, Ser144Ile,Glu146Ser, Glu146Thr, Glu146Val, Glu146Leu, Glu146Ile, Pro156Ala,Pro156Val, Pro156Ile, Gln158Thr, Gln158Val, Lys160Leu, Ser161Val,Ser161Ile, Lys172Ser, Lys172Ala, Lys172Val, Lys172Leu, Thr176Leu,Arg182Ala, Arg182Leu, Lys209Ala, and/or Lys209Val.
 5. A vaccine,comprising one or more of the modified influenza H5 haemagglutinins ofclaim 4, and a pharmaceutically acceptable carrier.
 6. A method ofimmunizing a subject against a H5 influenza virus infection, comprisingadministering to the subject at least one therapeutically effective doseof the vaccine of claim
 5. 7. A method of immunizing a subject against aH5 influenza virus infection, wherein at least a first and secondtherapeutically effective dose of a vaccine comprising one or more ofthe influenza H5 haemagglutinins of claim 4 and a pharmaceuticallyacceptable carrier are administered to a subject; wherein the first dosecomprises a first modified influenza H5 haemagglutinin according toclaim 4, and the second dose comprises a second modified influenza H5haemagglutinin according to claim 4, and wherein the first and secondinfluenza haemagglutinins are different.
 8. The method of claim 6,further comprising administration of an adjuvant.
 9. A method ofproducing therapeutic antibodies against conserved epitopes of influenzaH5 haemagglutinin comprising: (a) administration of one or more modifiedinfluenza H5 haemagglutinins of claim 4 to a subject; and (b) isolationof anti-H5 haemagglutinin antibodies.
 10. The modified influenza H5haemagglutinin of claim 4, in which at least three primary antigenicdeterminant residues of the primary immunodominant epitope are modifiedby replacement of at least one high LODrps amino acid with a low LODrpsamino acid, wherein the at least three modified primary antigenicresidues of amino acids 138-209 of SEQ ID NO:1 are selected from thegroup consisting of Ser141Thr, Ser141Ala, Ser141Val, Ser141Leu,Ser141Ile, Ser144Thr, Ser144Ala, Ser144Val, Ser144Leu, Ser144Ile,Glu146Ser, Glu146Thr, Glu146Ala, Glu146Val, Glu146Leu, Glu146Ile,Pro156Ala, Pro156Val, Pro156Leu, Pro156Ile, Gln158Ser, Gln158Thr,Gln158Ala, Gln158Val, Lys160Leu, Lys160Ile, Ser161Thr, Ser161Ala,Ser161Val, Ser161Leu, Ser161Ile, Lys172Ser, Lys172Thr, Lys172Ala,Lys172Val, Lys172Leu, Lys172Ile, Thr176Leu, Thr176Ile, Arg182Thr,Arg182Ala, Arg182Leu, Lys209Ser, Lys209Thr, Lys209Ala, Lys209Val,Lys209Leu, and/or Lys209Ile.
 11. A modified influenza H5 haemagglutinin,comprising amino acids 138-209 of SEQ ID NO:9.
 12. The method of claim1, further comprising modifying at least three of the primary antigenicdeterminant residues having a high LODrps value by replacing the atleast three of the primary antigenic determinant residues having a highLODrps value with an amino acid having a low LODrps value therebycreating a modified influenza H5 haemagglutinin with reducedantigenicity, wherein the at least three modified primary antigenicdeterminant residues of amino acids 138-209 of SEQ ID NO:1 are selectedfrom the group consisting of Ser141Thr, Ser141Ala, Ser141Val, Ser141Leu,Ser141Ile, Ser144Thr, Ser144Ala, Ser144Val, Ser144Leu, Ser144Ile,Glu146Ser, Glu146Thr, Glu146Ala, Glu146Val, Glu146Leu, Glu146Ile,Pro156Ala, Pro156Val, Pro156Leu, Pro156Ile, Gln158Ser, Gln158Thr,Gln158Ala, Gln158Val, Lys160Leu, Lys160Ile, Ser161Thr, Ser161Ala,Ser161Val, Ser161Leu, Ser161Ile, Lys172Ser, Lys172Thr, Lys172Ala,Lys172Val, Lys172Leu, Lys172Ile, Thr176Leu, Thr176Ile, Arg182Thr,Arg182Ala, Arg182Leu, Lys209Ser, Lys209Thr, Lys209Ala, Lys209Val,Lys209Leu, and/or Lys209Ile.
 13. A vaccine made by the method of claim12.
 14. The method of claim 7, further comprising administration of anadjuvant.